ClC Chloride Channels in Gram-Negative Bacteria and Its Role in the Acid Resistance Systems

Pathogenic bacteria that colonize the human intestinal tract have evolved strategies to overcome acidic conditions when they pass through the gastrointestinal tract. Amino acid-mediated acid resistance systems are effective survival strategies in a stomach that is full of amino acid substrate. The amino acid antiporter, amino acid decarboxylase, and ClC chloride antiporter are all engaged in these systems, and each one plays a role in protecting against or adapting to the acidic environment. The ClC chloride antiporter, a member of the ClC channel family, eliminates negatively charged intracellular chloride ions to avoid inner membrane hyperpolarization as an electrical shunt of the acid resistance system. In this review, we will discuss the structure and function of the prokaryotic ClC chloride antiporter of amino acid-mediated acid resistance system.

promotes cell survival [15]. Since Salmonella cannot survive with an internal pH of less than 5.5, these mechanisms are advantageous in increasing both the internal and external pH [16].
In extremely acidic conditions, the intracellular proton concentrations continuously increase due to the influx of HCl and the activity of ClC chloride channels. Bacterial cells must therefore evaluate how they consume or release these internal protons. For glucose-dependent AR1, the F o F 1 H + -translocating ATPase on the cell membrane causes the release of intracellular protons into the extracellular environment [17]. As a result, if the F o F 1 ATPase is defective, AR1 cannot function properly, whereas AR2 and 3 are unaffected regardless of the presence of ATPase [15]. During acid stress at pH 2.5, both AR2 and AR3 elevate the intercellular pH [15]. Moreover, at pH 2.5, the electrochemical gradient (Δψ) across the membrane was reversed by AR2 and AR3 to survive in the extremely acidic conditions. Reversal of membrane potential from inside negative to inside positive charge might be beneficial for E. coli to minimize excessive proton motive force generated during acid stress [15,18,19]. Simultaneously, the ClC channel exchanges internal protons with external chloride ions in opposite directions in order to balance positive electrical potential [20].
On the other hand, bacteria including E. coli convert unsaturated fatty acids into cyclopropane fatty acids, which are in part controlled by RpoS [21]. Because the unsaturated fatty acids contribute to internal membrane fluidity, decreasing membrane proton permeability prevents the accumulation of intracellular protons ahead of time. In conclusion, bacteria have both resistance and protection strategies against extremely acidic stress, which are similar to those of acidophiles thriving under acidic conditions.

The Conserved ClC Channel Family in Gram-Negative Bacteria
The ClC chloride channel family is a conserved protein group that is widely distributed from prokaryotes to human muscle cells [22]. Yet, the sequence similarities between kingdoms are quite low, ranging from 15 to 20% requires F o F 1 ATPase for acid resistance. When bacteria are challenged by extremely acidic conditions, levels of cyclic adenosine monophosphate (cAMP) drop, which otherwise bind to the receptor (CRP) and inhibit synthesis of RpoS. The decrease in cAMP levels thus enhances RpoS and promotes production of GadE. And, GadE enhances transcription of gadABC to control AR2. The right side panel represents the amino acid-dependent acid resistance systems including AR2, AR3, AR4, and AR5. The substrates enter through amino acid-product antiporters and are decarboxylated via the respective decarboxylases into positively charged products, which are then exported through the antiporters. During decarboxylation, protons are consumed by decarboxylases. Chloride ions, the counterions of hydrochloric acid, are exported by ClC chloride antiporters. The substrates and products are indicated on the bottom side of the panel.  [23]. Although overall similarities are low, chloride coordinating "hot spots" and key residues are highly conserved across the species and kingdoms ( Fig. 2A) [24]. Furthermore, the hydrophobicity patterns are strongly conserved in all known ClC channels. These ClC channel proteins share nine to twelve transmembrane helices and membrane topology [25,26].
In the phylogenetic tree of ClcA, Shigella flexneri is close to E. coli, which is expected to possess a similar XAR mechanism (Fig. 2B) [6,27]. However, despite sharing the sequence similarity, V. cholerae is very different from the other species on the ClcA phylogenetic tree. This might be due to that V. cholerae lacks XAR system and hence only a small number of bacteria survive at pH 2.5, making them less likely to colonize the intestine [5].

Functional Characterization of ClcA in the Amino Acid-Mediated AR System
In the amino acid-mediated AR systems, the ClC chloride channels exchange two internal chloride ions for one proton from the outside of the cell [28]. This transporter does more than just release excess ions in the electrogenic process. Because the amino acid antiporters export more positively charged products than the substrates (for examples, Glu to GABA + / Arg + to Agm 2+ ), transmembrane potential is shifted to inside negative. At the same time, chloride ions accumulate in the cytoplasm due to HCl ionization, resulting in inner membrane hyperpolarization [14]. In these circumstances, the ClC channels are used as electric shunts to counteract hyperpolarization.
The primary gene of the prokaryotic ClC channel family, clcA (also known as eriC or yadQ), controls the amino acid-mediated AR system to be functional, which is linked to cell survival [29]. Despite the fact that there is no impairment depending on the number or function of amino acid-substrate antiporters or amino acid decarboxylases, the rate of releasing decarboxylated products to extracellular solution from E. coli with a defective ClC channel is significantly lower than that of wild-type [14]. In the case of V. cholerae, the survival rate of the clcA knock-out strain decreased over time at pH 5 [30]. According to these findings, the prokaryotic ClC channel clcA gene controls the entire amino acid-mediated AR systems, and it affects the survival of bacterial pathogens in a wide range of acidic environments. Because ClcA influences the AR systems, its activity is dramatically reduced when the AR system is not required, such as neutral or basic pH conditions [14]. A V. cholerae strain deleting ClC channel showed sensitivity to acidic stress but unexpectedly exhibited increased intestinal colonization in infant mice model compared to control group [31]. Later, a search for in vivo repressed genes using a recombination-based in vivo expression technology (RIVET) implicates that clcA gene is required for acidic resistance in the stomach but its expression needs to be repressed in the lower gastrointestinal tract because its presence in the lower intestinal tract decreases V. cholerae survival fitness in infant mouse model [30].

Structure and Function in Prokaryotic ClC Chloride Antiporter
The ClC subclasses of the ClC family are split into two categories: transmembrane ion channels and antiporters [25]. The ClC channels and transporters have a similar overall structure [23]. Among them, prokaryotic ClC is an H + /Clantiporter that exports chloride ions outside of gram-negative bacteria such as E. coli, S. Typhimurium, and V. cholerae.
Prokaryotic ClC chloride antiporter is a double-pore homodimer that is fundamentally different from a common cation channel, which is usually a single-pore protein with four-or five-fold symmetry [32,33]. In the case of S. Typhimurium, ClC antiporter has 18 helices, the majority of which are tilted and embedded in the membrane [34]. Due to its symmetry, each subunit's pore is twisted rather than aligned straight [35]. Also, the pore lining is composed of four sequence segments [36].
The prokaryotic ClC chloride antiporter undergoes distinctive conformational changes during Cltransport cycle, releasing two chloride ions and one proton on opposite direction [37]. Chloride antiporter alternates opening and closing of two pores in the extracellular and intracellular sides in concert with the conformational changes. Major steps are following: 1) Two pores open outwards and bind to chloride ions, but both the intracellular pores are closed. 2) Two pores open inwards and release chloride ions, but extracellular pores are closed. At this time, key residues (E203 in E. coli) for proton transport face cytoplasm and accessible for proton transport. In between these two steps, there are transitory conformational changes toward inward or outward opening of the two pores.
Key residues were identified in crystallographic studies of ClC antiporter, which are widely conserved and function as selectivity filters by coordinating chloride ion and proton (Fig. 3) [38]. Glutamate side chains directly control the passage of chloride ion depending proton binding status, thus coupling chloride ion movement to proton transport. E148 residue functions as a gate by blocking the movement of chloride ions in a deprotonated state but it also involves the release of chloride ions in a protonated state [39,40]. In addition to E148, E203 residue affects chloride-proton antiport via protonation or deprotonation in the transmembrane region [41]. When the extracellular pores open and E148 is protonated, E203 is buried in the protein interior and inhibits protonation [33]. Although E148 and E203 participate in the proton transport pathway, the distance between the two residues is as far as 15 Å, which is not enough to transport the proton with a single movement [42]. For that reason, the hydroxyl group of Y445, which is located between two glutamates, directly coordinates with the central chloride ion in the ClC antiporter. Because the electrophilic aromatic ring of Y445 serves as a proton donor, chloride ions are easily accessible [34]. Mutations in Y445 render the protein inactive or weaken proton coupling, resulting in a slower rate of proton transport [33,43].
There are four key residues (S107, E148, E203, and Y445) that directly coordinate chloride ion transport (Fig. 3B). S107, similarly to Y445, coordinates with oxygen in the amino acid side-chain and directly binds to chloride ions, affecting ion conductance in the pore [34]. When bound to the polar functional group, chloride ions interact with additional hydrophobic amino acid side chains and are released into the extracellular space, resulting in conformational change.  Fig. 2. Red-colored residues (S107, E148, E203, and Y445) are key residues in the selectivity filter and chloride ion binding in ClcA. Blue-colored residues are additional selectivity filter in ClcA. (B) A magnified view of four key residues involved in glutamate gates and chloride ion binding. Green spheres represent chloride ions in the ClC transporter. The homodimer is visualized using PyMOL. To distinguish each monomer, one subunit of the homodimer is visualized more transparently.

Another ClC Channel Family Protein, ClcB
ClcB (also known as mriT and ynfJ), a member of the bacterial ClC chloride channel family, has crucial roles in controlling intracellular chloride ion concentration and enhancing cell viability in the extremely acidic condition. Although clcB has not been studied as thoroughly as clcA, a double knock-out mutant of clcA and clcB exhibited a much lower survival in acidic conditions than wild-type and single knock-out mutants [14]. ClcB is also conserved within gram-negative bacteria and is considered as a chloride ion transporter (Fig. 4). As previously discussed, V. cholerae is extremely sensitive to pH 2.5 [5], probably due to lacking XAR system. Whether the absence of ClcB in V. cholerae could contribute to extreme sensitivity to pH 2.5 needs to be addressed.
Analyzing the homologues of ClcA and ClcB suggests that ClcB contains key residues similar to those found in ClcA. Because the glutamate gate in ClcB is located similarly to those in ClcA, the selectivity filter and chloride binding sites in ClcB appear to function in a similar manner to those in ClcA. Further research is required to determine the precise mechanism of ClcB in chloride transport.

Conclusions
In bacteria, acid resistance systems including XAR and ATR have been commonly used as survival strategies in extracellular acidic environments such as the human gastrointestinal tract. Among the AR systems, AR2, AR3, AR4, and AR5 are classified as amino acid-mediated AR systems. In the amino acid-mediated AR systems, amino acid antiporter, amino acid decarboxylase, and ClC chloride antiporter are three major components. Amino acid antiporters transport negative charged substrates (Glu, Arg, Lys, and Orn) into the cytoplasm and export positively charged products (GABA, Agm, Cad, and Put), respectively. Amino acid decarboxylases consume protons generated by the influx of HCl as they convert amino acids into positively charged products. Finally, ClC chloride antiporters prevent inner membrane hyperpolarization by transporting chloride ions to the extracellular solution. In this process, the membrane potential of neutralophilic bacteria changes from negative inside to positive charge inside, resulting in an electrochemical gradient similar to acidophiles. This characteristic resolves electrophysiological problems caused by excess intracellular protons generated in extremely acidic conditions. The ClC chloride antiporter plays a significant role in the amino acid-mediated AR system. This transporters are found in a wide range of organisms, ranging from bacteria to humans, and its key residues are highly conserved. Prokaryotic clcA genes encode ClC transporters that control the AR system in acidic conditions. In the ClC antiporter, key residues in the ion selectivity filter are two glutamate residues that coordinate chloride ion binding in the center of the ClC antiporter. This selectivity filter releases two chloride ions and binds to one proton from the outside simultaneously. This process promotes survival of bacteria from acidic conditions by preventing membrane hyperpolarization, thus allowing bacteria to colonize the human intestinal tract.

Author Contributions
E.-J.L. conceptualize the review and wrote the manuscript; M.K. wrote the manuscript; N.C. analyze the data and wrote the manuscript; E.C. revise the manuscript.